Monoclonal antibodies

A general representation of the methods used to produce monoclonal antibodies.

Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell.

Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine. When used as medications, the non-proprietary drug name ends in -mab (see "Nomenclature of monoclonal antibodies").

Discovery

The idea of a "magic bullet" was first proposed by Paul Ehrlich, who, at the beginning of the 20th century, postulated that, if a compound could be made that selectively targeted a disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity. He and Élie Metchnikoff received the 1908 Nobel Prize for Physiology or Medicine for this work, which led to an effective syphilis treatment by 1910.

In the 1970s, the B-cell cancer multiple myeloma was known, and it was understood that these cancerous B-cells all produce a single type of antibody (a paraprotein). This was used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given antigen.

Production of monoclonal antibodies involving human–mouse hybrid cells was described by Jerrold Schwaber in 1973^[1] and remains widely cited among those using human-derived hybridomas,^[2] but claims to priority have been controversial. A science history paper on the subject gave some credit to Schwaber for inventing a technique that was widely cited, but stopped short of suggesting that he had been cheated.^[3] The invention was conceived by George Pieczenik, with John Sedat, Elizabeth Blackburn's husband, as a witness and reduced to practice by Cotton and Milstein, and then by Kohler and Milstein. Georges Köhler, César Milstein, and Niels Kaj Jerne in 1975;^[4] who shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery. The key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies, come up with a technique to fuse these cells with healthy antibody-producing B-cells, and be able to select for the successfully fused cells.

In 1988, Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies,^[5] removing the reactions that many monoclonal antibodies caused in some patients.

Production

Researchers looking at slides of cultures of cells that make monoclonal antibodies. These are grown in a lab and the researchers are analyzing the products to select the most promising of them.

Monoclonal antibodies can be grown in unlimited quantities in the bottles shown in this picture.

Technician hand-filling wells with a liquid for a research test. This test involves preparation of cultures in which hybrids are grown in large quantities to produce desired antibody. This is effected by fusing myeloma cell and mouse lymphocyte to form a hybrid cell (hybridoma).

Lab technician bathing prepared slides in a solution. This technician prepares slides of monoclonal antibodies for researchers. The cells shown are labeling human breast cancer.

Hybridoma cell production

Further information: Hybridoma technology

Monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen. However, recent advances have allowed the use of rabbit B-cells to form a Rabbit Hybridoma. Polyethylene glycol is used to fuse adjacent plasma membranes, but the success rate is low so a selective medium in which only fused cells can grow is used. This is because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids. The absence of HGPRT is not a problem for these cells unless the de novo purine synthesis pathway is also disrupted. By exposing cells to aminopterin (a folic acid analogue, which inhibits dihydrofolate reductase, DHFR), they are unable to use the de novo pathway and become fully auxotrophic for nucleic acids requiring supplementation to survive.

The selective culture medium is called HAT medium because it contains hypoxanthine, aminopterin, and thymidine. This medium is selective for fused (hybridoma) cells. Unfused myeloma cells cannot grow because they lack HGPRT, and thus cannot replicate their DNA. Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid cells, referred to as hybridomas, are able to grow indefinitely in the media because the spleen cell partner supplies HGPRT and the myeloma partner has traits that make it immortal (as it is a cancer cell).

This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen (with a test such as ELISA or Antigen Microarray Assay) or immuno-dot blot. The most productive and stable clone is then selected for future use.

The hybridomas can be grown indefinitely in a suitable cell culture medium.They can also be injected into mice (in the peritoneal cavity, surrounding the gut). There, they produce tumors secreting an antibody-rich fluid called ascites fluid.

The medium must be enriched during in-vitro selection to further favour hybridoma growth. This can be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as briclone. Culture-medium conditioned by macrophages can also be used. Production in cell culture is usually preferred as the ascites technique is painful to the animal. Where alternate techniques exist, this method (ascites) is considered unethical.

Purification of monoclonal antibodies

After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. The contaminants in the cell culture sample would consist primarily of media components such as growth factors, hormones, and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic acids, and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as a result, endotoxins that are secreted by the bacteria. Depending on the complexity of the media required in cell culture, and thus the contaminants in question, one method (in vivo or in vitro) may be preferable to the other.

The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by centrifugation followed by filtration with a 0.45 µm filter. These large particles can cause a phenomenon called membrane fouling in later purification steps. In addition, the concentration of product in the sample may not be sufficient, especially in cases where the desired antibody is one produced by a low-secreting cell line. The sample is therefore condensed by ultrafiltration or dialysis.

Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography.^[6] Either cation exchange chromatography is used at a low enough pH that the desired antibody binds to the column while anions flow through, or anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. Various proteins can also be separated out along with the anions based on their isoelectric point (pI). For example, albumin has a pI of 4.8, which is significantly lower than that of most monoclonal antibodies, which have a pI of 6.1. In other words, at a given pH, the average charge of albumin molecules is likely to be more negative. Transferrin, on the other hand, has a pI of 5.9, so it cannot easily be separated out by this method. A difference in pI of at least 1 is necessary for a good separation.

Transferrin can instead be removed by size exclusion chromatography. The advantage of this purification method is that it is one of the more reliable chromatography techniques. Since we are dealing with proteins, properties such as charge and affinity are not consistent and vary with pH as molecules are protonated and deprotonated, while size stays relatively constant. Nonetheless, it has drawbacks such as low resolution, low capacity and low elution times.

A much quicker, single-step method of separation is Protein A/G affinity chromatography. The antibody selectively binds to Protein A/G, so a high level of purity (generally >80%) is obtained. However, this method may be problematic for antibodies that are easily damaged, as harsh conditions are generally used. A low pH can break the bonds to remove the antibody from the column. In addition to possibly affecting the product, low pH can cause Protein A/G itself to leak off the column and appear in the eluted sample. Gentle elution buffer systems that employ high salt concentrations are also available to avoid exposing sensitive antibodies to low pH. Cost is also an important consideration with this method because immobilized Protein A/G is a more expensive resin.

To achieve maximum purity in a single step, affinity purification can be performed, using the antigen to provide exquisite specificity for the antibody. In this method, the antigen used to generate the antibody is covalently attached to an agarose support. If the antigen is a peptide, it is commonly synthesized with a terminal cysteine, which allows selective attachment to a carrier protein, such as KLH during development and to the support for purification. The antibody-containing media is then incubated with the immobilized antigen, either in batch or as the antibody is passed through a column, where it selectively binds and can be retained while impurities are washed away. An elution with a low pH buffer or a more gentle, high salt elution buffer is then used to recover purified antibody from the support.

To further select for antibodies, the antibodies can be precipitated out using sodium sulfate or ammonium sulfate. Antibodies precipitate at low concentrations of the salt, while most other proteins precipitate at higher concentrations. The appropriate level of salt is added in order to achieve the best separation. Excess salt must then be removed by a desalting method such as dialysis.

The final purity can be analyzed using a chromatogram. Any impurities will produce peaks, and the volume under the peak indicates the amount of the impurity. Alternatively, gel electrophoresis and capillary electrophoresis can be carried out. Impurities will produce bands of varying intensity, depending on how much of the impurity is present.

Recombinant

The production of recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected.^[7] The phage antibody libraries are a variant of the phage antigen libraries first invented by George Pieczenik [1] These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications.^[8] Fermentation chambers have been used to produce these antibodies on a large scale.

Chimeric antibodies

Main article: Chimeric antibodies

Early on, a major problem for the therapeutic use of monoclonal antibodies in medicine was that initial methods used to produce them yielded mouse, not human antibodies. While structurally similar, differences between the two are sufficient to invoke an immune response occurred when murine monoclonal antibodies were injected into humans and resulted in their rapid removal from the blood, systemic inflammatory effects, and the production of human anti-mouse antibodies (HAMA).^{[citation needed]}

In an effort to overcome this obstacle, approaches using recombinant DNA have been explored since the late 1980s. In one approach, mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells, and the expression of this chimeric DNA through cell culture yielded partially-mouse, partially-human monoclonal antibody. For this product, the descriptive terms "chimeric" and "humanised" monoclonal antibody have been used to reflect the combination of mouse and human DNA sources used in the recombinant process.^[9]

'Fully' human monoclonal antibodies

Ever since the discovery that monoclonal antibodies could be generated in-vitro, scientists have targeted the creation of 'fully' human antibodies to avoid some of the side effects of humanised and chimeric antibodies. Two successful approaches were identified — phage display-generated antibodies and mice genetically engineered to produce more human-like antibodies.

One of the most successful commercial organisations behind therapeutic monoclonal antibodies was Cambridge Antibody Technology (CAT). Scientists at CAT demonstrated that phage display could be used such that variable antibody domains could be expressed on filamentous phage antibodies. This was reported in a key Nature publication.^[10]

Other significant publications include:

• Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G (December 1991). "By-passing immunization. Human antibodies from V-gene libraries displayed on phage". J. Mol. Biol. 222 (3): 581–597. doi:10.1016/0022-2836(91)90498-U. PMID 1748994. http://linkinghub.elsevier.com/retrieve/pii/0022-2836(91)90498-U. 
• Carmen S, Jermutus L (July 2002). "Concepts in antibody phage display". Brief Funct Genomic Proteomic 1 (2): 189–203. doi:10.1093/bfgp/1.2.189. PMID 15239904. http://bfgp.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=15239904. 

CAT developed their display technologies further into several, patented antibody discovery/functional genomics tools, which were named Proximol^TM[11] and ProAb^TM. ProAb was announced in December 1997^[12] and involved highthroughput screening of antibody libraries against diseased and non-diseased tissue, whilst Proximol used a free radical enzymatic reaction to label molecules in proximity to a given protein.^[13][14]

Genetically engineered mice, so called transgenic mice, can be modified to produce human antibodies,^[15] and this has been exploited by a number of commercial organisations:

• Medarex — who market their UltiMab platform^[16]
• Abgenix — who marketed their Xenomouse technology. Abgenix were acquired in April 2006 by Amgen.^[17]
• Regeneron's VelocImmune technology.^[18]

Monoclonal antibodies have been generated and approved to treat: cancer, cardiovascular disease, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infection (see monoclonal antibody therapy).

In August 2006 the Pharmaceutical Research and Manufacturers of America reported that U.S. companies had 160 different monoclonal antibodies in clinical trials or awaiting approval by the Food and Drug Administration.^[19]

Applications

Diagnostic tests

Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance. The Western blot test and immuno dot blot tests detect the protein on a membrane. They are also very useful in immunohistochemistry, which detect antigen in fixed tissue sections and immunofluorescence test, which detect the substance in a frozen tissue section or in live cells...

Therapeutic treatment

Main article: Monoclonal antibody therapy

Cancer treatment

One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cell-specific antigens and induce an immunological response against the target cancer cell. Such mAb could also be modified for delivery of a toxin, radioisotope, cytokine or other active conjugate; it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell. In fact, every intact antibody can bind to cell receptors or other proteins with its Fc region.

Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment.^[20]

The illustration below shows all these possibilities:

MAbs approved by the FDA include^[21]

• Bevacizumab
• Cetuximab
• Panitumumab
• Trastuzumab

Autoimmune diseases

Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which are effective in rheumatoid arthritis, Crohn's disease and ulcerative Colitis by their ability to bind to and inhibit TNF-α.^[22] Basiliximab and daclizumab inhibit IL-2 on activated T cells and thereby help prevent acute rejection of kidney transplants.^[22] Omalizumab inhibits human immunoglobulin E (IgE) and is useful in moderate-to-severe allergic asthma.

Examples

For a more comprehensive list, see List of monoclonal antibodies.

List of FDA approved therapeutic antibodies is found in Monoclonal antibody therapy

Below are examples of clinically important monoclonal antibodies.

Main category	Type	Application	Mechanism/Target	Mode
Anti- inflammatory	infliximab[22]	rheumatoid arthritis Crohn's disease Ulcerative Colitis	inhibits TNF-α	chimeric
	adalimumab	rheumatoid arthritis Crohn's disease Ulcerative Colitis	inhibits TNF-α	human
	basiliximab[22]	Acute rejection of kidney transplants	inhibits IL-2 on activated T cells	chimeric
	daclizumab[22]	Acute rejection of kidney transplants	inhibits IL-2 on activated T cells	humanized
	omalizumab	moderate-to-severe allergic asthma	inhibits human immunoglobulin E (IgE)	humanized
Anti-cancer	gemtuzumab[22]	relapsed acute myeloid leukaemia	targets myeloid cell surface antigen CD33 on leukemia cells	humanized
	alemtuzumab[22]	B cell leukemia	targets an antigen CD52 on T- and B-lymphocytes	humanized
	rituximab[22]	non-Hodgkin's lymphoma	targets phosphoprotein CD20 on B lymphocytes	chimeric
	trastuzumab	breast cancer with HER2/neu overexpression	targets the HER2/neu (erbB2) receptor	humanized
	nimotuzumab	Approved in squamous cell carcinomas, Glioma Clinical trials for other indications underway	EGFR inhibitor	Humanized
	cetuximab	Approved in squamous cell carcinomas, colorectal carcinoma	EGFR inhibitor	Chimeric
	bevacizumab	Anti-angiogenic cancer therapy	inhibits VEGF	humanized
Other	palivizumab[22]	RSV infections in children	inhibits an RSV fusion (F) protein	humanized
	abciximab[22]	Prevent coagulation in coronary angioplasty	inhibits the receptor GpIIb/IIIa on platelets	chimeric

See also

• Antibody mimetic
• Immunotoxins, which sometimes use monoclonal antibodies as the targeting mechanism
• List of monoclonal antibodies
• Monoclonal antibody therapy
• Nomenclature of monoclonal antibodies
• Polyclonal antibodies
• Queen Mab Small mythological figure symbolizing hope (popular culture, used as biotech pun).

References

1. ^ Schwaber, J.; Cohen, E. P. (1973). "Human x mouse somatic cell hybrid clone secreting immunoglobulins of both parental types". Nature 244 (5416): 444–447. Bibcode 1973Natur.244..444S. doi:10.1038/244444a0. PMID 4200460.  edit
2. ^ Science Citation Index
3. ^ Cambrosio, A.; Keating, P. (1992). "Between fact and technique: the beginnings of hybridoma technology". Journal of the History of Biology 25 (2): 175–230. doi:10.1007/BF00162840. PMID 11623041.  edit
4. ^ Köhler, G.; Milstein, C. (1975). "Continuous cultures of fused cells secreting antibody of predefined specificity". Nature 256 (5517): 495–497. Bibcode 1975Natur.256..495K. doi:10.1038/256495a0. PMID 1172191.  edit
5. ^ Riechmann L, Clark M, Waldmann H, Winter G (March 1988). "Reshaping human antibodies for therapy". Nature 332 (6162): 323–327. doi:10.1038/332323a0. PMID 3127726. 
6. ^ Vlasek J, Ionescu R (2008). "Hetergeneity of Monoclonal Antibodies Revealed by Charge-Sensitive Methods". Current Pharmaceutical Biotechnology 9 (6): 468–481. doi:10.2174/138920108786786402. PMID 19075686. 
7. ^ Siegel DL (2002). "Recombinant monoclonal antibody technology". Transfusion clinique et biologique : journal de la Société française de transfusion sanguine 9 (1): 15–22. PMID 11889896. 
8. ^ Schmitz U, Versmold A, Kaufmann P, Frank HG (2000). "Phage display: a molecular tool for the generation of antibodies—a review". Placenta 21 (Suppl A): S106–S112. doi:10.1053/plac.1999.0511. PMID 10831134. 
9. ^ Helen E. Chadd and Steven M. Chamow, “Therapeutic antibody expression technology,” Current Opinion in Biotechnology 12, no. 2 (April 1, 2001): 188-194.
10. ^ McCafferty, J.; Griffiths, A.; Winter, G.; Chiswell, D. (1990). "Phage antibodies: filamentous phage displaying antibody variable domains". Nature 348 (6301): 552–554. Bibcode 1990Natur.348..552M. doi:10.1038/348552a0. PMID 2247164.  edit
11. ^ Osbourn JK (2002). "Proximity-guided (ProxiMol) antibody selection". Methods Mol. Biol. 178: 201–5. PMID 11968489. 
12. ^ http://www.ukbusinesspark.co.uk/cay92125.htm
13. ^ http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T76-3WBNNJS-6&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=960781456&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=eacedcd4604792172fc18143a590a454
14. ^ http://www.chidb.com/newsarticles/issue3_1.ASP
15. ^ Lonberg N, Huszar D (1995). "Human antibodies from transgenic mice". Int. Rev. Immunol. 13 (1): 65–93. doi:10.3109/08830189509061738. PMID 7494109. 
16. ^ http://www.medarex.com/Development/UltiMAb.htm
17. ^ http://wwwext.amgen.com/media/media_pr_detail.jsp?year=2006&releaseID=837754
18. ^ http://www.regeneron.com/velocimmune.html
19. ^ PhRMA Reports Identifies More than 400 Biotech Drugs in Development. Pharmaceutical Technology, August 24, 2006. Retrieved 2006-09-04.
20. ^ Modified from Carter P: Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 2001;1:118-129
21. ^ Takimoto CH, Calvo E. "Principles of Oncologic Pharmacotherapy" in Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ (Eds) Cancer Management: A Multidisciplinary Approach. 11 ed. 2008.
22. ^ ^{a b c d e f g h i j} Rang, H. P. (2003). Pharmacology. Edinburgh: Churchill Livingstone. pp. 241, for the examples infliximab, basiliximab, abciximab, daclizumab, palivusamab, gemtuzumab, alemtuzumab and rituximab, and mechanism and mode. ISBN 0-443-07145-4. 

External links

• Monoclonal Antibodies Animation (Flash Required)
• Monoclonal Antibodies, from John W. Kimball's online biology textbook
• MeSH Monoclonal+antibodies